Effect of Tris, MOPS, and phosphate buffers on the

hydrolysis of polyethylene terephthalate films by polyester

hydrolases

rio-Ferrari, Markus Barth,
Juliane Schmidt, Ren Wei, Thorsten Oeser, Matheus Regis Belisa
Johannes Then and Wolfgang Zimmermann
Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Germany

Keywords The enzymatic degradation of polyethylene terephthalate (PET) occurs at

3-(N-morpholino)propanesulfonic acid; mild reaction conditions and may find applications in environmentally
biocatalysis; inhibition; polyester hydrolase;
friendly plastic waste recycling processes. The hydrolytic activity of the
polyethylene terephthalate; Tris
homologous polyester hydrolases LC cutinase (LCC) from a compost
Correspondence metagenome and TfCut2 from Thermobifida fusca KW3 against PET films
R. Wei and W. Zimmermann, Department was strongly influenced by the reaction medium buffers tris(hydrox-
of Microbiology and Bioprocess Technology, ymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid
Institute of Biochemistry, Leipzig University, (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis
Johannisallee 21-23, D-04103 Leipzig, rate of PET films in 0.2 M Tris, while the rate of TfCut2 was 2.1-fold lower
Germany
at this buffer concentration. At a Tris concentration of 1 M, the hydrolysis
Fax: +49 341 97 36798
rate of LCC decreased by more than 90% and of TfCut2 by about 80%.
Tel: +49 341 97 36781
E-mails: wei@uni-leipzig.de; In 0.2 M MOPS or sodium phosphate buffer, no significant differences in
wolfgang.zimmermann@uni-leipzig.de the maximum initial hydrolysis rates of PET films by both enzymes were
detected. When the concentration of MOPS was increased to 1 M, the
(Received 10 May 2016, revised 21 June hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2
2016, accepted 24 June 2016) remained low compared to the increasing hydrolysis rates observed at
higher concentrations of sodium phosphate buffer. In contrast, the activity
doi:10.1002/2211-5463.12097
of LCC did not change at different concentrations of this buffer. An inhi-
bition study suggested a competitive inhibition of TfCut2 and LCC by Tris
and MOPS. Molecular docking showed that Tris and MOPS interfered
with the binding of the polymeric substrate in a groove located at the
protein surface. A comparison of the Ki values and the average binding
energies indicated MOPS as the stronger inhibitor of the both enzymes.

Polyethylene terephthalate is a synthetic aromatic widespread use of synthetic polyesters such as PET
polyester composed of ethylene glycol and terephthalic contributes to growing amounts of postconsumer plas-
acid (TPA) [1]. Because of its versatile properties PET tic wastes. The related environmental pollution prob-
is used in many products such as textile fibers or bev- lems require efficient recycling processes. The
erage bottles. The high strength, low weight, low per- biocatalytic degradation of PET offers a less energy-
meability of gases, and its resistance to many consuming and environmentally friendly method than
chemicals make PET an excellent packaging material conventional recycling processes [4,5]. Hydrolytic activ-
for a wide range of products [2]. In 2013, about 13% ity against PET has been shown for lipases [1,5–7],
of all packaging materials were made of PET [3]. The carboxylesterases [6–9], and cutinases [1,6,7,10].

Abbreviations
BHET, bis-(2-hydroxyethyl) terephthalate; LCC, LC cutinase; MHET, mono-(2-hydroxyethyl) terephthalate; MOPS, 3-(N-morpholino)
propanesulfonic acid; PET, polyethylene terephthalate; TPA, terephthalic acid.

FEBS Open Bio 6 (2016) 919–927 ª 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 919
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
Effect of buffers on polyester hydrolases J. Schmidt et al.

Several polyester hydrolases from fungi [1], actino- RP-HPLC. An inhibitory effect of Tris and MOPS
mycetes [5,11–19], and from a compost metagenome buffer on the two hydrolases was investigated experi-
[10,20] have been reported. mentally as well as by docking experiments.
The type and ionic strength of buffers can influence
the enzymatic activity in aqueous reaction systems. Materials and methods
The effect of ions and inorganic salts on the solubility
of proteins has been reported more than 120 years Genes, enzymes and chemicals
ago, when Hofmeister proposed a series of cations and
anions according to their ability to precipitate hen egg The synthetic LCC gene construct with adapted codon
white [21]. It has been shown that Hofmeister effects usage for Escherichia coli was obtained from GeneArt
can also influence the catalytic activity of enzymes. Gene Synthesis (Life Technologies GmbH, Darmstadt,
The ionic strength as well as the pH of a buffer system Germany). Amorphous PET films (250 lm thickness) were
affect the activity and structural features of enzymes. purchased from Goodfellow GmbH (Bad Nauheim, Ger-
many, product number 029-198-54). FastDigest restriction
The activity of immobilized Candida rugosa and Rhizopus
enzymes were purchased from Life Technologies GmbH.
oryzae lipases was influenced by the ionic strength of
All other chemicals were obtained from Carl Roth GmbH
the buffer and was highest in a mixture of 0.25 M
+ Co. KG (Karlsruhe, Germany) and Gruessing GmbH
MOPS and sodium phosphate [22]. In another study,
Analytica (Filsum, Germany) at the highest purity
both buffers as well as salt ions showed specific
available.
Hofmeister effects on the enzymatic activity of the
C. rugosa lipase [23]. Weak and strong electrolytes
strongly influenced the enzymatic activity of the lipase. Cloning and expression of the polyester
While sulfate ions increased the activity, chloride hydrolase genes
behaved neutrally and thiocyanate strongly decreased The synthetic LCC gene construct without the secretion sig-
it. The influence of inorganic salts and ions on the sta- nal peptide (ENA: LN879395) was amplified using the pri-
bility and activity of alkaline phosphatase from calf mers LCC-FW (50 -TTTTGGATCCGTCTAACCCGTACC
intestine has also been shown [24]. The activating and AGCGTG-30 ) and LCC-RV (50 -TTTTGAATTCCCCTGG
stabilizing effect of different inorganic salts correlated CAGTGACGGTTGTT G-30 ). The gene for TfCut2 (ENA:
well with their kosmotropic and chaotropic properties FR727681) without signal peptide was amplified from
proposed in the Hofmeister series. An influence of the T. fusca KW3 genomic DNA using the primers TfCut2-
ionic strength of the buffer system on the hydrolysis of FW (50 -TTTTTTGGATCCGGCCAACCCCTACGAGCGC
PET films in a membrane reactor by the polyester -30 ) and TfCut2-RV (50 -TTTTTGAATTCGGGTAGAACG
hydrolase TfCut2 has also been reported [25]. The GGCAGGTGG AGC-30 ) (the restriction sites for BamHI
highest initial hydrolysis rates were obtained in or EcoRI are underlined for each primer). The amplified
Na2HPO4 buffer at a concentration of 0.7 M. genes were digested with FastDigest BamHI and EcoRI
An inhibition of the activity of aminopeptidases and and ligated into the BamHI and EcoRI restriction sites of
aminotransferases [26–28], cholinesterases [29], and dif- the vector pET-20b(+). The final pET-20b(+) construct
ferent carbohydrate hydrolases [30–35] by Tris has containing the ligated genes, pelB leader sequence, and
His6 tag was cloned into E. coli BL21 (DE3). The expres-
been reported. Most of these studies indicated a com-
sion and purification of the recombinant hydrolases was
petitive inhibition of the enzymes by this buffer.
carried out as described previously [38]. After purification,
MOPS has been shown to inhibit the activity of bovine
the enzymes were concentrated and the buffer was changed
adrenal tyrosine hydroxylase [36]. The related sulfonic
to 10 mM Tris–HCl (pH 8.0 at 60 °C) using Amicon Ultra
acid buffer 4-morpholinoethanesulfonic acid (MES)
Centrifugal Filter Units (Merck KGaA, Darmstadt,
also inhibited the metallo-b-lactamase from Germany).
Bacteroides fragilis [37].
Previously, we have examined the effect of the ionic
strength of HEPES, MOPS, PIPES, Tris, and sodium Hydrolysis of PET films by LCC and TfCut2
phosphate buffers on the enzymatic hydrolysis of PET Polyethylene terephthalate films of 9 cm2 (about 150 mg)
films in a membrane reactor [25]. In this study, we were added to reaction vials containing 0.1–2.8 lgcm2 of
investigate the effect of these buffer types on the purified LCC or TfCut2 and 0.1–1 M Tris, sodium phos-
hydrolysis of PET by two polyester hydrolases in fur- phate or MOPS buffer (pH 8.0) in a total volume of
ther detail. The degradation of amorphous PET films 1.8 mL. The pH of the buffers was adjusted at 60 °C using
by TfCut2 and LCC at different buffer concentrations HCl for Tris and NaOH for MOPS buffer. The reaction
was monitored by measuring the released products by vials were incubated at 60 °C on a thermo shaker

920 FEBS Open Bio 6 (2016) 919–927 ª 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
J. Schmidt et al. Effect of buffers on polyester hydrolases

(1000 rpm) for 1 h. Released hydrolysis products were a 2.1-fold higher rate than TfCut2 (Fig. 1A). In 0.2 M
quantified by RP-HPLC [39]. The sum of the released sol- MOPS and sodium phosphate, both enzymes did not
uble products—TPA, mono-(2-hydroxyethyl) terephthalate significantly differ in their maximum hydrolysis rates
(MHET), and bis-(2-hydroxyethyl) terephthalate (BHET) (40–60 lMh1) (Fig. 1B,C).
was used to determine the initial hydrolysis rate. All initial
rates were determined at least in triplicate.
Effect of buffer concentrations on the hydrolysis
of PET films by TfCut2 and LCC
Inhibition of LCC and TfCut2 by Tris and MOPS
The effect of the buffer concentration on the hydroly-
Polyethylene terephthalate films of 1–9 cm2 (about sis rate of LCC and TfCut2 against PET films is
20–150 mg) were added to reaction vials containing purified shown in Fig. 2. LCC and TfCut2 were employed in
LCC (1 lg) or TfCut2 (5 lg) and 0.2 M sodium phosphate concentrations corresponding to their maximum initial
buffer (pH 8.0) in a total volume of 1.8 mL. Tris (0.2–
hydrolysis rates (see Fig. 1). The activity of TfCut2
0.4 M, pH 8.0) and MOPS (0.05–0.3 M, pH 8.0) were added
and LCC depended on the concentration of the buf-
to the reaction mixture. The vials were incubated at 60 °C
fers. The activity of TfCut2 in MOPS buffer was low
on a thermo shaker (1000 rpm) for 1 h. Released hydroly-
in the range from 0.1 to 1 M. In 1 M Tris buffer, its
sis products were quantified by RP-HPLC [39].

A 160
Molecular docking of Tris and MOPS to LCC and
Initial hydrolysis rate
140
TfCut2 120
[μM•h–1] 100
The crystal structures of TfCut2 (PDB: 4CG1) [40] and 80
LCC (PDB: 4EB0) [20] were used for the docking experi- 60
ments with AutoDock Vina [41]. The 3D conformer struc- 40
tures of neutral Tris (CID 6503) [42] and protonated 20
MOPS (CID 2723950) [43] were obtained from the Open 0
Chemistry Database [44]. The ligand structure of a PET 0 0.5 1 1.5 2 2.5 3
model substrate (2PET) consisting of two units of the [E] [μg•cm–2]
monomer ethylene terephthalate was built with MOE
(Chemical Computing Group, Montreal, Canada). Auto- B 160
Initial hydrolysis rate

DockTools (v1.5.6) (Molecular Graphics Laboratory, 140

Department of Molecular Biology, The Scripps Research 120

100
[μM•h–1]

Institute, La Jolla, CA, USA) was used to prepare the files

80
and select the search space. PyMOL 1.1r1 [45] was used for
60
the visualization of the results. The main binding sites were
40
selected and the average binding energy was calculated.
20
0
0 0.5 1 1.5 2 2.5 3
Results
[E] [μg•cm–2]

Effect of enzyme concentration and buffer C 160

composition on the hydrolysis of PET films by
Initial hydrolysis rate

140
TfCut2 and LCC 120
100
[μM•h–1]

When the hydrolysis of PET films was performed at

80
different concentrations of TfCut2 and LCC in Tris– 60
HCl, MOPS, and sodium phosphate buffers (0.2 M, 40
pH 8.0), the initial hydrolysis rates increased with 20
increasing enzyme concentrations until a maximum 0
was reached and then leveled off or decreased at 0 0.5 1 1.5 2 2.5 3

higher enzyme concentrations (Fig. 1). The maximum [E] [μg•cm–2]

initial hydrolysis rates were reached with lower LCC
Fig. 1. Initial hydrolysis rates of PET films (9 cm2) as a function of
concentrations (0.1–0.3 lgcm2) compared to TfCut2 the concentration of TfCut2 (dashed line) and LCC (solid line) in (A)
(0.6 lgcm2) in all three buffers. LCC showed the Tris; (B) MOPS; (C) sodium phosphate (0.2 M, pH 8.0). Error bars
highest activity in Tris–HCl buffer (146.6 lMh1) with indicate the standard deviation of triplicate determinations.

FEBS Open Bio 6 (2016) 919–927 ª 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 921
Effect of buffers on polyester hydrolases J. Schmidt et al.

A A 0.6
1600
Initial hydrolysis rate
[μM•h–1•μg–1•cm2]
1400

1/v0 [h•μM–1]
1200 0.4
1000
800 0.2
600
400
200 0
–0.2 0 0.2 0.4 0.6 0.8 1 1.2
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
–0.2 1/[S] [cm–2]
Buffer concentration [M]
B B 0.6
Initial hydrolysis rate

1600
[μM•h–1•μg–1•cm2]

1400
1200 0.4

1/v0 [h•μM–1]
1000
800
0.2
600
400
200 0
0 –0.2 0 0.2 0.4 0.6 0.8 1 1.2
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Buffer concentration [M] –0.2
1/[S] [cm–2]
C
1600
Initial hydrolysis rate

Fig. 3. Double reciprocal plots of initial hydrolysis rates of PET

[μM•h–1•μg–1•cm2]

1400
1200 films versus substrate concentration for LCC (A) and TfCut2 (B) at
1000 different concentrations of Tris: ● 0 M, ▲ 0.2 M, and ♦ 0.4 M.
800
600
400 8.0) containing 0.2 and 0.4 M of Tris for both enzymes
200
0 or 0.1 and 0.3 M of MOPS for LCC. Since the hydrol-
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 ysis rates of PET by TfCut2 at MOPS concentrations
Buffer concentration [M] of 0.1–1 M were very low, buffer concentrations in the
range from 0.05–0.075 M were used. LCC and TfCut2
Fig. 2. Initial hydrolysis rates of PET films (9 cm2) of LCC (light
were employed in concentrations corresponding to
bars) and TfCut2 (dark bars) as a function of buffer concentration
of (A) Tris, (B) MOPS, and (C) sodium phosphate (pH 8.0). In each
their maximum initial hydrolysis rates (see Fig. 1).
buffer, LCC and TfCut2 were employed in concentrations The Lineweaver–Burk plots indicated an inhibition
corresponding to their maximum initial hydrolysis rates (see Fig. 1). of both enzymes by Tris and MOPS (Figs 3 and 4).
Error bars indicate the standard deviation of triplicate All curves showed common intercepts located mainly
determinations. in the first square of the plots suggesting a competitive
type of inhibition of LCC and TfCut2 by Tris and
activity decreased by 80% compared to 0.1 M Tris buf- MOPS. A similar result has been reported previously
fer. In contrast, the hydrolysis rate was about 10-fold indicating a competitive inhibition of TfCut2 by the
higher at 1 M sodium phosphate compared to 0.1 M. PET hydrolysis products MHET and BHET [39].
LCC showed a different response to changing buffer The corresponding Ki values (Table 1) for both buf-
concentrations. While the hydrolysis rates were unaf- fers were determined by replotting the slopes calculated
fected within 0.1–1 M of the phosphate buffer, in 1 M from the Lineweaver–Burk plots against the concentra-
Tris and MOPS buffers its activity decreased by more tion of Tris and MOPS [46,47]. The results showed that
than 90% compared to 0.1 M. MOPS is the stronger inhibitor of both enzymes with
significantly lower Ki values compared to Tris.
Inhibition of the hydrolysis of PET by LCC and
TfCut2 in the presence of Tris and MOPS Molecular docking of Tris and MOPS
The low hydrolysis rates detected with Tris and MOPS The molecular docking experiments performed for
buffers suggested an inhibitory effect on the hydrolysis LCC and TfCut2 with the two inhibitors, Tris and
of PET by LCC and TfCut2. The inhibitory effect of MOPS, revealed several binding sites on the surface of
the buffers was investigated by performing the hydrol- the two enzymes. An interaction of the inhibitors with
ysis reaction in 0.2 M sodium phosphate buffer (pH specific amino acid residues could not be confirmed

922 FEBS Open Bio 6 (2016) 919–927 ª 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
J. Schmidt et al. Effect of buffers on polyester hydrolases

A 0.6 2.8 kcalmol1 bound to TfCut2. The average bind-

ing energies indicated a higher affinity of MOPS for
1/v0 [h•μM–1]

0.4 both enzymes and confirmed the lower Ki values of

MOPS (Table 1).
0.2

0 Discussion
–0.2 0 0.2 0.4 0.6 0.8 1 1.2

–0.2 1/[S] [cm–2] In this study, the effect of MOPS, Tris and sodium
phosphate buffers on the enzymatic activity of the
B 0.6 polyester hydrolases LCC and TfCut2 was investi-
gated. The determination of the initial hydrolysis rates
0.4 showed that maximum values were obtained at lower
1/v0 [h•μM–1]

concentrations of LCC than of TfCut2 indicating a

0.2 higher activity of LCC against the PET film. In 0.2 M
Tris LCC showed a 2.1-fold higher maximum initial
0
–0.2 0 0.2 0.4 0.6 0.8 1 1.2 hydrolysis rate than TfCut2. In contrast, both enzymes
revealed no significant difference in their maximum
–0.2 1/[S] [cm–2]
hydrolysis rates in 0.2 M MOPS or sodium phosphate
Fig. 4. Double reciprocal plots of initial hydrolysis rates of PET buffer. At increasing concentrations of Tris and MOPS
films versus substrate concentration for LCC (A) and TfCut2 (B) at buffer, the initial PET hydrolysis rates decreased or
different concentrations of MOPS: (A) ● 0 M, ▲ 0.2 M, and were constantly low for LCC and TfCut2. An inverse
♦ 0.4 M and (B) ● 0 M, ▲ 0.05 M, and ♦ 0.075 M. effect was observed with sodium phosphate buffer.
TfCut2 showed an increased initial hydrolysis rate with
Table 1. Inhibition of the hydrolysis of PET films by LCC and
increasing sodium phosphate concentrations while
TfCut2 through Tris and MOPS. The Ki values were determined by LCC showed constantly high initial hydrolysis rates at
replotting the slopes calculated from the Lineweaver–Burk plots all tested concentrations. The kosmotropic phosphate
against the concentration of Tris and MOPS. anions possibly stabilized LCC and TfCut2 resulting
in higher hydrolysis rates of the PET films. The precip-
Inhibitor Enzyme Ki [M]
itating (salting out) and solubilizing (salting in) proper-
Tris LCC 0.24 ties of anions have been explained by different water
TfCut2 0.44 adsorbing effects of salts [21]. Ions are thereby classi-
MOPS LCC 0.17
fied according to their ability to form or break water
TfCut2 0.08
structures. Kosmotropic (precipitating) ions stabilize
proteins and support the formation of polar water
(Fig. 5). The main binding site of Tris and MOPS was structures, whereas chaotropic (solubilizing) ions break
detected near the catalytic triad inside the substrate- the water structures and destabilize proteins [49–52].
binding groove at the surface of LCC and TfCut2 [48]. Such a stabilizing effect has been reported previously
A second minor binding site of Tris was located next for a tetrameric maize leaf phosphoenolpyruvate car-
to the substrate-binding pocket of LCC (Fig. 5A). This boyxlase [53]. The authors suggested that kosmotropic
site is assumed to be inside an extended region of the anions such as HPO2 4 stabilized the enzyme most
substrate-binding pocket of LCC when compared to effectively by their water-structuring effects and by
the substrate-binding regions of the polyester hydro- increasing the surface tension. The observed stabilizing
lases from Thermobifida cellulosilytica DSM44535 [16]. effect also depended on the concentration of the kos-
A similar minor binding site of MOPS was observed motropic salts. HPO2 4 significantly improved the sta-
in TfCut2 (Fig. 5D). The docking experiments with bility and activity of an endoxylanase from Bacillus sp.
Tris were carried out in its protonated and neutral [54]. By increasing the concentration of K2HPO4, an
state since both are present at pH 8.0. The same bind- increased Tm value and an increase in xylanase activity
ing areas were obtained for both states and only the was observed. The authors suggested that the activity
results of neutral Tris are shown in Fig. 5. increase and the stabilization of the enzyme by
A determination of the average binding energies K2HPO4 was due to a conformational change caused
showed for MOPS 3.7 kcalmol1 bound to LCC by the phosphate anion. Supporting these findings, an
and 3.5 kcalmol1 bound to TfCut2. For Tris, activating effect of sodium phosphate buffer was also
3.1 kcalmol1 were obtained bound to LCC and observed for LCC and TfCut2 (Fig. 2C).

FEBS Open Bio 6 (2016) 919–927 ª 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 923
Effect of buffers on polyester hydrolases J. Schmidt et al.

Fig. 5. Docking of Tris and MOPS to LCC

and TfCut2. An overlay of multiple binding
modes of the inhibitors is presented. The
structures of the enzymes are shown in
red with the catalytic triad highlighted as
blue sticks and Tris and MOPS as yellow
sticks. (A) docking of Tris (neutral) to LCC;
(B) docking of Tris (neutral) to TfCut2; (C)
docking of MOPS to LCC; (D) docking of
MOPS to TfCut2. The docking was
performed with AutoDock Vina [41].

While TfCut2 showed the highest initial hydrolysis enzymes (Table 1). When comparing the initial hydrol-
rate in sodium phosphate buffer at a concentration of ysis rates of PET films by TfCut2 and LCC in 0.1–1 M
> 0.7 M, the hydrolysis rate of LCC was independent Tris and MOPS buffers, MOPS also caused a decrease
from the concentration of the sodium phosphate buffer in the hydrolysis rates at lower concentrations than
in a range from 0.1 to 1 M. Since TPA is released dur- Tris (Fig. 2). An inhibition of cholinesterases by Tris
ing the hydrolysis of PET, a buffer of high molarity or has been already reported in the 1960s [29]. The
strength is required to maintain the pH of the reaction authors showed a competitive inhibition by Tris with
medium [55]. The ionic strength of the buffer has been inhibition constants of 13–14 mM. A competitive inhi-
shown previously to influence the hydrolysis of PET bition of an a-amylase from Bacillus licheniformis by
films by TfCut2 in an ultrafiltration membrane reactor Tris with a Ki of 13.12 mM has also been reported
[25]. The enzyme showed a 3.8-fold higher initial [46]. Accompanying docking studies indicated a high
hydrolysis rate in 0.5 M Na2HPO4 than in 0.5 M Tris binding potential of Tris at the active site of the
buffer. Confirming our results, the highest hydrolysis enzyme. The catalytic residues Asp 174 and Glu 200
rate was also obtained at a sodium phosphate concen- of a psychrophilic a-amylase from Alteromonas halo-
tration of 0.7 M. The effect of the Na2HPO4 buffer on planctis also showed a strong interaction with the
the hydrolytic activity of TfCut2 was attributed to the amino group of Tris [56]. In addition, the hydroxyl
high ionic strength of the buffer [25]. At 0.5 M, the groups of Tris were found to form hydrogen bonds
Na2HPO4 buffer has an ionic strength of 3 M, while with the three catalytic amino acids of the enzyme.
the ionic strength of Tris buffer with this concentra- The Ki values obtained in this study for the inhibition
tion is 0.07 M. When the ionic strength of Tris buffer of LCC and TfCut2 by Tris (Table 1) are higher than
was increased to 2 M, a 2.4-fold higher hydrolysis rate those described in other reports before, suggesting a
of TfCut2 was observed. weaker inhibiting effect than for other enzymes.
In contrast to sodium phosphate buffer, higher con- Only few reports are available about an inhibitory
centrations of Tris and MOPS resulted in a reduction effect of MOPS on enzyme activity. MOPS has been
of the hydrolytic activity of LCC and TfCu2 against shown to reduce the activity of bovine adrenal tyrosine
PET suggesting an inhibitory effect of the buffers hydroxylase by 40% [36]. A metallo-b-lactamase from
(Fig. 2A,B). The double reciprocal plots suggested a Bacteroides fragilis was inhibited by MES which is
competitive inhibition. A comparison of the Ki values similar to MOPS a sulfonic acid buffer [37]. The crys-
indicated MOPS as a stronger inhibitor for both tal structure of a complex of the enzyme with MES

924 FEBS Open Bio 6 (2016) 919–927 ª 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
J. Schmidt et al. Effect of buffers on polyester hydrolases

suggested an interaction of MES with the active site of References

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